DESCRIPTION: The folding of large RNA molecules can be approached as a hierarchical process in which pre-formed substructures assemble into the final, complex 3-dimensional structure. Important and little understood RNA building units are the multi-helix junction loops, the simplest of which are the three-way junctions (3WJ). Such structures occur frequently in large RNAs; a better knowledge of their folding rules would significantly improve our ability to predict and model the three-dimensional structures of a large variety of RNAs. In vitro selection and evolution methodologies (hereafter referred to as SELEX) will be employed to explore the molecular interactions which stabilize and define the structures of RNA 3WJ. Dr. Leontis will apply SELEX to generate, select, and amplify a library of RNA molecules capable of forming 3WJ structures, in which nucleotide positions surrounding the junction site are randomized. The most compact and thermally stable species in the RNA pool will be selected by thermal gradient gel electrophoresis and amplified reiteratively. After sufficient cycles have been completed, such that a large percentage of the pool exhibits the desired physical properties, a sampling of RNA sequences from the pool will be cloned, sequenced, and chemically and enzymatically probed to characterize the atomic interactions which stabilize the structures. Molecular modeling techniques will be employed to construct 3-dimensional atomic models of the structures of representative molecules using the probing data as a guide. Modeling studies will be carried out in collaboration with Dr. Eric Westhof, a leader in the field of RNA structure modeling. The thermal stabilities of these molecules will be measured using UV-monitored thermal analysis ("UV melting") and isothermal titration calorimetry to provide thermodynamic parameters to correlate with the structural models. Further experimental verification for the 3-dimensional structures will be sought by attempting to crystallize interesting sequences. If crystals are obtained, crystallographic analysis will be pursued. Solution structures will be characterized using multi-dimensional/multi-nuclear NMR spectroscopy.